Central nervous system noradrenergic signaling modulates attention, mood, arousal, learning and memory. Specifically, norepinephrine reuptake is implicated in the pathology of major depression, posttraumatic stress disorder and attention deficit disorder. Therapeutic agents for the treatment of depression inhibit norepinephrine transporter (NET) and serotonin transporter (SERT) activities, elevating the concentration of these transmitters in the synaptic cleft. Antidepressants that inhibit NET, such as desipramine, are effective for the treatment of depression. Biogenic amine transporters are also biological targets for drugs of abuse, such as cocaine and amphetamine. The work described in this research proposal investigates substrate translocation, in which we develop novel methodology for measuring NET, SERT and dopamine transporter (DAT) activity. The fluorescent-based uptake assay described in Specific Aim I can be used as a nonisotopic method for high-throughput drug screening probing for compounds that interact with NET, SERT or DAT. The studies outlined in Specific Aim II examine the correlation between substrate and charge movement. Information garnered from these studies, can help assess the contribution of neurotransmitter transporters to synaptic transmission. The advancement in methodology enables the analysis NET activity in individual neurons, thus providing insight into endogenous NET function. These data may also provide a better understanding of therapeutic agents and drugs of abuse.